ABSTRACT ? Characterization Unit The Characterization Unit will be responsible for omic and multiscale image analyses of pairs of biopsies taken pre- and on/post treatment from patients with metastatic breast and prostate cancer undergoing treatment with current generation pathway and immune checkpoint targeted treatments. The paired biopsies represent sensitive and resistant lesions and comparative analyses of these enable discovery of mechanisms of resistance. Twenty pairs of samples each from three treatment cohorts will be analyzed. Specifically, (a) hormone-receptor positive breast cancer (HRBC) undergoing treatment with a CDK4/6 inhibitor in combination with endocrine therapy, (b) triple negative breast cancer (TNBC) undergoing treatment with a PARP inhibitor and an immunomodulatory agent, and (c) castration resistant prostate cancer (CRPC) undergoing treatment with enzalutamide. Aim 1 will analyze OCT frozen biopsies using Topographic Single Cell Sequencing (TSCS) to cells isolated from spatially defined regions to enable analysis of genomic copy number changes, mutations, and indels and Single-cell Combinatorial Indexing ATAC-seq (sci-ATAC-seq) to elucidate chromatin accessibility in single cells. The cells analyzed using sci-ATAC-seq will be computationally mapped to cyclic immunofluorescence (cycIF) stained sec- tions based on predicted gene expression patterns. Aim 2 will analyze formalin-fixed paraffin embedded (FFPE) sections using multiplex immunohistochemistry (mIHC) and cycIF to assess the tumor and stromal cell compo- sition and the molecular states thereof. In addition, next generation cycIF using oligonucleotide labeled antibod- ies will be developed to enable high dimension cycIF staining and imaging using both conventional and super resolution fluorescence microscopy. Aim 3 will analyze paraformaldehyde fixed, resin embedded (PFRE) speci- mens using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to identify ultrastructural changes in 2D images and targeted 3D volumes with 4-nm resolution. In addition, work in this aim will assess the utility of Serial Block Face Imaging SEM (SBFI) to define 3D ultrastructural changes in larger volumes, but at lower res- olution.